M-MLV Reverse transcriptase

Reaction system
Template RNA (Total RNA 10 pg-5 μg, Poly(A) RNA 10 pg-500 ng); Oligo(dT)18 (50 μM) or Random Primer Mix (50 ng/μL) 1 μL; dNTP Mix (10 mM each) 1 μL; RNase Inhibitor (40 U/μL) 1 μL; Reverse transcriptase (200 U/μL) 1 μL; Reaction buffer (250 mM tri-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl₂, 50 mM DTT) 4 μL; Nuclease-free Water Up to 20 μL.
Reaction procedure
(1) 25°C for 5 minutes; (2) 50°C for 45 minutes; (3) 85°C 2 min.
(A) Add this incubation step if Random Primer Mix is used; This step is omitted when using Oligo(dT)18 or Gene Specific Primer.
(B) If the template has a complex secondary structure or a high GC region, the reaction temperature can be raised to 55°C, which is helpful to increase the yield.
(C) Appropriately extending the reverse transcription time can help obtain longer cDNA (>5 kb).
Precautions
1) To prevent RNase contamination, please keep the experimental area clean and wear clean gloves and masks during operation. Used in the experiment.
All consumables such as centrifuge tubes and pipette tips must be RNase-free.
2) The cDNA product can be stored at -20°C or -80°C or immediately used for PCR (qPCR) reactions.
3) The volume of cDNA products used in PCR (qPCR) reactions is recommended not to exceed one-tenth of the PCR reaction volume.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.